Synaptotagmin 1 likely acts as a Ca²⁺ sensor in neurotransmitter release by Ca²⁺-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca²⁺ affinity of synaptotagmin 1 and in the Ca²⁺ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca²⁺ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca²⁺ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca²⁺ sensitivity of release is dictated by the apparent Ca²⁺ affinity of synaptotagmin 1 in both directions, and that Ca²⁺ binding to both C2 domains contributes to Ca²⁺ triggering of release.